Reversal of the phenotype of transformed cells by the transcription factor IRF-1

Abstract: The invention relates to the use of IRF-1 or nucleic acids containing the coding information for IRF-1 for the reversal of the phenotype of transformed cells. Nucleic acids coding for IRF-1 can be used as tumor suppressor genes in the gene therapy of cancer.

Description: The present invention relates to the use of the transcription factor interferon regulatory factor-1 (IRF-1) or nucleic acid molecules containing the coding information for IRF-1 for the manufacture of a pharmaceutical composition to reverse the phenotype of tumor cells or cells which show a transformed phenotype. Interferon regulatory factor-1 (IRF-1), a transcriptional activator, and IRF-2, its antagonistic repressor, have been identified as regulators of type I interferon (IFN) and IFN-inducible genes. It has been shown previously that IRF-1 manifests anti-oncogenic activity in NIH 3T3 cells which are transformed by overexpression of IRF-2. In fact, when the IRF-2 gene was overexpressed in NIH 3T3 cells, the cells became transformed and displayed enhanced tumorigenicity in nude mice. However, this transformed phenotype was reversed by concomitant overexpression of the IRF-1 gene (Harada et al ., Science 259 , 971-974, 1993). It has been shown that the human IRF-1 gene maps to chromosome 5q31.1, a region frequently deleted in patients with leukemia or preleukemic myelodysplastic syndromes (Willman et al ., Science 259 , 968-971, 1993). In the present invention, it has surprisingly been found that the reversal of the transformed phenotype by IRF-1 is not restricted to cells transformed by its natural antagonist, IRF-2, but can also be achieved in cells expressing oncogenes. So, this invention makes it possible to revert the transformed phenotype in tumor cells more generally. IRF-1 and nucleic acids coding for IRF-1 can be applied in the therapy of cancer, especially in gene therapy. Thus, the present invention is related to the use of a nucleic acid containing the coding information for interferon regulatory factor 1 (IRF-1) or a biologically active variant or fragment thereof for the manufacture of a pharmaceutical composition for the treatment of a disease treatable by reversal of the phenotype of transformed cells, which express the oncogene c.myc or fos B . Transformed cells or tumor cells are cells characterized by significant changes in their growth properties as compared to normal cells (Darnell et al ., Molecular Cell Biology, Scientific American Books, Inc., New York, 1986, pp. 1039-1046). It is further preferred that said cells are characterized by lack or shortage of tumor suppressor gene expression. Advantageously, said nucleic acid is incorporated into an expression vector. This can be, for example, a viral vector, preferably a retroviral vector, e.g. pDG. A preferred embodiment of the present invention is the gene transfer of a nucleic acid containing the coding information for interferon regulatory factor 1 (IRF-1) or a biologically active variant or fragment thereof into transformed or tumor cells. As a gene transfer vehicle, an appropriate retrovirus can be used. However, in another preferred embodiment said nucleic acid molecule is complexed with a conjugate, said conjugate consisting of an endosomolytical agent and a DNA binding agent (see below). Preferably, said endosomolytical agent is an inactivated adenovirus and said DNA-binding agent is polylysine. The present invention is further related to the use of a nucleic acid containing the coding information for IRF-1 or a biologically active variant or fragment thereof for the manufacture of a pharmaceutical composition for the therapy of cancer wherein the cancer cells of said cancer express the oncogene c-myc or fos B, especially when said pharmaceutical composition is suited for the gene therapy of cancer. Said nucleic acid may also contain regulatory sequences. In one preferred embodiment, the coding information for IRF-1 is incorporated in an nucleic acid molecule which is complexed with a conjugate, said conjugate consisting of an endosomolytical agent and an DNA binding agent. Preferably, said endosomolytical agent is an inactivated adenovirus or adenoviral particles. Preferably, said DNA binding agent is polylysine. In another aspect, the present invention is related to the use of IRF-1 polypeptide or a biologically active variant or fragment thereof for the manufacture of a pharmaceutical composition for the therapy of cancer wherein the cancer cells of said cancer express the oncogene c-myc or fos B . The IRF-1 of the present invention comes preferably from the same species as the transforemd or cancer cells which are to be treated. If human cells are to be treated, the IRF-1 is preferably human IRF-1. The experts knows from the art how to produce nucleic acid molecules containing the coding information for IRF-1, for example IRF-1 from mouse (Miyamoto et al., Cell 54 , 903-913, 1988) or man (Maruyama et al., Nucl . Acids Res. 17 , 3292, 1989; EP 0359998). He further knows how to generate nucleic acid molecules coding for variants or fragments and methods to test whether such variants or fragments still exhibit the biological activity of the parent molecule. Thus, such variants and fragments are also encompassed by the present invention. The skilled expert further knows methods from the art how to integrate the coding information for IRF-1 into nucleic acids capable of expressing the gene, and to introduce such nucleic acid molecules into cells in a way that the IRF-1 gene is expressed in the target cells. More detailed examples how the present invention can be carried out are described below. The inventor of the present invention attempted to revert the phenotype of rat Rat 1 cells transformed by either c-myc gene or fosB gene. These two genes are well known oncogenes and their properties have been well studied (Shiroki et al ., Mol. Cell. Biol. 6 , 4379-4386, 1986; Nakabeppu et al ., Mol . Cell. Biol . 13 , 4157-4166, 1993). A retrovirus, pDGIRF1, which expresses murine IRF-1 upon infection, was previously constructed (Harada et al ., Science 259 , 971-974, 1993). The c - myc -transformed 3Y1 cell or fosB -transformed Rat-1A cell, termed RmycY1 and Rat-1A(FosB), respectively, both show anchorage-independent growth and enhanced tumorigenicity in rat or nude mice (Shiroki et al ., Mol. Cell. Biol . 6 , 4379-4386, 1986; Nakabeppu et al., Mol . Cell. Biol. 13 , 4157-4166, 1993). 5 x 10 5 of each cells were infected by the pDGIRF-1 virus at the multiplicity of infection (m.o.i.) of 10, using exactly the method employed by Harada et al ., and subsequently limiting diluted to obtain single clones. After 1 or 2 weeks, these cell clones have lost the transformed phenotype. In fact, these virally infected cells have changed their morphology which is indistinguishable from the original Rat 1 cells (Figures 1,2), and lost their tumorigenic potential in nude mice (Table 1). In contrast, no such change was observed by infecting similarly the control retrovirus pGD (Figures 1,2). Thus, these observations clearly indicate the broad function of IRF-1 as tumor suppressor in cells transformed by at least two different oncogenes. We also observed that the levels of the oncogene messenger RNAs expressed in these reverted cells remained essentially the same as the original, transformed cells. Therefore, the observed phenotypic reversion of the cells as the result of IRF-1 gene expression is not due to the inhibition of the promoters used for the oncogene expression. The results suggest that some of the cancer cells may be suppressed either completely, as shown here, or partially for their phenotype. Hence, the present invention may be very important for the future development of IRF-1 gene as promising tumor suppressor gene for gene therapy of cancer, especially for cancers which contain activated oncogenes or are characterized by lack or shortage of tumor suppressor gene expression. It can be also emphasized that the IRF-1 gene transfer can be achieved not only by the presently employed retrovirus but also by other means, i.e. the present invention is not restricted to the use of the IRF-1-expressing retrovirus, rather, it points to the general importance of IRF-1 gene transfer in the suppression of tumor cells. Reviews of gene transfer systems and clinical protocols which can be adapted for use of the present invention can be found in the literature (Morgan et Anderson, Ann . Rev. Biochem . 62 , 191-217, 1993; Roemer et Friedmann, Eur. J. Biochem. 208 , 211-225, 1992). A gene transfer system which can be used advantageously in connection with the present invention has been developed recently (Curiel et al ., Proc . Natl. Acad. Sci. USA 88 , 8850-8854, 1991; Curiel et al ., Am. J. Respir . Cell Mol . Biol . 6 , 247-252, 1992a; Zatloukal et al ., Ann . New York Acad. Sci. 660 , 136-153, 1992; Cotten et al ., Proc . Natl. Acad Sci. USA 89 , 6094-6098, 1992; Wagner et al ., Proc . Natl. Acad. Sci. USA 89 , 6099-6103, 1992; Curiel et al., Human Gene Therapy 3 , 147-154, 1992b). It is based on the property of adenoviruses to discharge the content of endosomes after having been incorporated by receptor-mediated endocytosis. The use of adenoviruses enhances the efficiency of gene transfer because it circumvents the lysosomal degradation of internalised DNA. The adenovirus can be modified by conjugation with polylysine. Since polylysine is cationic under physiological conditions, the resulting conjugate can complex DNA, for example DNA coding for IRF-1, by electrostatic interactions. The adenovirus-polylysine conjugates can be used to complex DNA together with transferrin-polylysine conjugates, resulting in ternary adenovirus-polylysine/transferrin-polylysine/DNA complexes (Wagner et al., loc . cit., 1992). Such complexes can bind to receptors specific for adenoviruses and/or transferrin and are internalized afterwards. After endocytosis, the adenovirus causes the break of the endosomal membrane and its content is discharged into the cytoplasm. The IRF-1-coding DNA can enter the cell nucleus, where IRF-1 is expressed mainly by episomally localized DNA. Growth properties of RmycY1 and Rat-1A(FosB) cells overexpressing IRF-1 Cells Growth efficiency (%) in methylcellulose gel Tumorigenicity Tumors per injection Latency (weeks) RmycY1 28, 26 6/6 3 to 4 3Y1 0, 0 0/6 myc-IRF1-1 <1, <1 0/2 myc-IRF1-2 <1, <1 0/2 myc-IRF1-3 <1, <1 0/2 myc-IRF1-4 <1, <1 0/2 myc-C-1 32, 34 2/2 3 to 4 myc-C-2 30, 28 2/2 3 to 4 Rat-1A(FosB) 24, 28 4/4 2 to 4 Rat-1A 0, 0 0/4 Fos B-IRF1-1 <1, <1 0/2 Fos B-IRF1-2 <1, <1 0/2 Fos B-IRF1-3 <1, <1 0/2 Fos B-IRF1-4 <1, <1 0/2 Fos B-C-1 26, 25 2/2 3 to 4 Fos B-C-2 18, 22 2/2 3 to 4 Brief description of the drawings Figure 1: Phenotypically reverted c- myc -transformed 3Y1 cells following retroviral introduction of IRF-1 . The cells were all photographed (magnification, x100) in Dulbecco modified Eagle medium containing 10% fetal calf serum. 3Y1-c-myc; c-myc-transformed 3Y1 cells (RmycY1), 3Y1; untransformed rat 3Y1 cells, myc-IRF1-1 and myc-IRF1-2; pGDIRF1 infected RmycY1 cell clones, myc-C-1 and myc-C-2; control virus pGD (Harada et al ., Science 259 , 971-974, 1993) infected RmycY1 cell clones. Figure 2: Phenotypically reverted fosB transformed Rat-1A cells following retroviral introduction of IRF-1 . The cells were all photographed (magnification, x100) in Dulbecco modified Eagle medium containing 10% fetal calf serum. Rat-1A-FosB; fosB transformed Rat-1A cells (Rat-1A(FosB)), Rat-1A; untransformed Rat-1A cells, Fos B-IRF1-1 and Fos B-IRF1-2; pGDIRF1 infected Rat-1A(FosB) cell clones, Fos B-C-1 and Fos B-C-2; pGD infected Rat-1A(FosB) cell clones. Example A retrovirus, pDGIRF1, which expresses murine IRF-1 upon infection, was constructed as previously described (Harada et al ., Science 259, 971-974, 1993). For this purpose, mouse IRF-1 cDNA (Miyamoto et al ., Cell 54 , 903-913, 1988) was inserted into the pGD vector (Daley et al ., Science 247 , 824, 1990). The DNA constructs were transfected into ψ2 cells (Mann et al ., Cell 33 , 153, 1983), which subsequently released into the culture medium a high titer (∼ 10 6 colony-forming units per milliliter) of virus, as assayed by the ability to confer neo resistance to NIH 3T3 cells. Expression of the IRF-1 protein was confirmed by gel-shift analysis. The c-myc -transformed 3Y1 cell or fosB -transformed Rat-1A cell, termed RmycY1 and Rat-1A(FosB), respectively, both show anchorage-independent growth and enhanced tumorigenicity in rat or nude mice (Shiroki et al ., Mol. Cell. Biol . 6 , 4379-4386, 1986; Nakabeppu et al., Mol . Cell . Biol . 13 , 4157-4166, 1993). 5 x 10 5 of each cells were infected by the pDGIRF-1 virus at the multiplicity of infection (m.o.i.) of 10, using exactly the method employed by Harada et al ., and subsequently limiting diluted to obtain single clones. Morphologic investigations and tumorigenicity tests (nude mice model) were performed by standard procedures. After 1 or 2 weeks, these cell clones have lost the transformed phenotype.

Claims: Use of a nucleic acid containing the coding information for interferon regulatory factor 1 (IRF-1) or a biologically active variant or fragment thereof for the manufacture of a pharmaceutical composition for the treatment of a disease treatable by reversal of the phenotype of cells which express the oncogene c-myc or fosB. Use of claim 1, wherein said cells are characterized by lack or shortage of tumor suppressor gene expression. Use of claim 1 or 2, wherein said nucleic acid is incorporated into a vector. Use of claim 3, wherein said vector is an expression vector. Use of claim 3, wherein said vector is a viral vector. Use of claim 1 or 2, wherein said nucleic acid is incorporated into a gene transfer vehicle. Use of claim 6, wherein said gene transfer vehicle is a retrovirus. Use of claim 7, wherein said retrovirus is pDGIRF-1. Use of claim 6, wherein said nucleic acid is complexed with a conjugate, said conjugate consisting of an endosomolytical agent and a DNA-binding agent. Use of claim 9, wherein said endosomolytical agent is an inactivated adenovirus. Use of claim 9, wherein said DNA-binding agent is polylysine. Use of a nucleic acid containing the coding information for IRF-1 or a biologically active variant or fragment thereof for the manufacture of a pharmaceutical composition for the therapy of cancer wherein the cancer cells of said cancer express the oncogene c-myc or fosB. Use of claim 12, wherein said pharmaceutical composition is for the gene therapy of cancer wherein the cancer cells of said cancer express the oncogene c-myc or fosB. Use of claim 12 or 13, wherein said nucleic acid is incorporated into a gene transfer vehicle. Use of claim 14, wherein said gene transfer vehicle is a virus. Use of claim 15, wherein said virus is a retrovirus. Use of claim 16, wherein said retrovirus is pDGIRF-1. Use of claim 12 or 13, wherein said nucleic acid is complexed with a conjugate, said conjugate consisting of an endosomolytical agent and a DNA-binding agent. Use of claim 18, wherein said endosomolytical agent is an inactivated adenovirus. Use of claim 18, wherein said DNA-binding agent is polylysine. Use of any one of claims 9 to 20, wherein the cancer cells are characterized by lack or shortage of tumor suppressor gene expression. Use of IRF-1 or a biologically active variant or fragment thereof for the manufacture of a pharmaceutical composition for the therapy of cancer wherein the cancer cells of said cancer express the oncogene c-myc or fosB.

Inventors: TADATSUGU TANIGUCHI, TADATSUGU, TANIGUCHI
Applicants: BOEHRINGER INGELHEIM INT, BOEHRINGER INGELHEIM INTERNATIONAL GMBH
Application year: 1994
Main classification: A61K31
IPC codes: A61K48, A61K38, C07K14, A61K47